Urinary Nucleosides Research Articles

Mid-level data fusion strategy based on urinary nucleosides SERS spectra and blood CEA levels for enhanced preoperative detection of lymph node metastasis in colorectal cancer

BackgroundPreoperative prediction of lymph node metastasis (LNM) plays a crucial role in the treatment and prognosis of colorectal cancer (CRC). The traditional histopathological examination is invasive and time-consuming, providing pathological features only postoperatively. Preoperative serum carcinoembryonic antigen (CEA) is strongly correlated with postoperative LN status. However, the detection accuracy of LNM based on a single preoperative CEA level is low. Therefore, developing a more powerful and sensitive diagnostic tool would be of great clinical value for improving the accurate preoperative prediction of LNM in CRC patients. ResultsThis study aimed to develop a mid-level fusion approach using urinary nucleosides Raman spectra and blood CEA data to enhance the preoperative discrimination of CRC patients with and without LNM. Surface-enhanced Raman scattering (SERS) spectra of urinary modified nucleosides, isolated by affinity chromatography, were first acquired from 48 patients with LNM and 49 patients without LNM. The principal component analysis (PCA) scores obtained from the SERS spectra were then combined with preoperative blood CEA values to create a fused data array. The discriminant accuracy based on either dataset alone or the fused data was evaluated using three machine learning algorithms: linear discriminant analysis, k-nearest neighbors, and support vector machine. Results showed that the fused data could discriminate between the two groups with an accuracy of up to 91 %, outperforming SERS alone (86 %) and CEA alone (69 %). SignificanceTo our knowledge, this is the first report of mid-level data fusion of urinary nucleosides SERS spectra with blood CEA levels for the preoperative prediction of LNM in CRC. This work demonstrates that the mid-level data fusion strategy aided by SVM algorithm can greatly improve the preoperative prediction accuracy of LNM. This is crucial for therapeutic decision-making and prognostic assessment in CRC.

Magnetic graphene oxide grafted boronic acid-decorated metal-organic frameworks for selective enrichment of cis-diol-containing nucleosides

In this study, a novel magnetic graphene oxide grafted boronic acid-decorated metal-organic frameworks (Fe3O4@GO@BA-MOF) composite was developed for the selective enrichment of cis-diol-containing nucleosides. It is noteworthy that the amino-functionalized Fe3O4 was prepared by polyethyleneimine (PEI) as modifier, resulting in excellent durability and stability of magnetic nanoparticles under different environment, as well as introducing rich active functional groups for post-modification. GO with large surface area and abundant multi-active group was covalently bonded on amino-rich Fe3O4 as interlayer to facilitate the growth of BA-MOF with high boronic acid grafting density for selective capture of nucleosides. Due to the multi-interactions, especially the boronate affinity as well as the synergistic effect, the Fe3O4@GO@BA-MOF composite exhibited superior selectivity and adsorption capacity to nucleosides. Under the optimal conditions, an efficient magnetic solid-phase extraction-high-performance liquid chromatography technique was established using Fe3O4@GO@BA-MOF composite for the purpose of extracting and detecting nucleosides in urine, with recoveries ranging from 90.69 % to 110.36 %, relative standard deviations below 8.5 % and detection limits ranging from 0.004 to 0.010 μg mL−1. This study offers a new approach for preparation of high grafting density of boronic acid-decorated magnetic MOF, which is promising in selective enrichment of cis-diol-containing compounds in biological analysis.

Exploring urinary modified nucleosides as biomarkers for diabetic retinopathy: Development and validation of a ultra performance liquid chromatography-tandem mass spectrometry method

The dynamic modification of RNA plays a crucial role in biological regulation and is strongly linked to human disease development and progression. Notably, modified nucleosides in urine have shown promising potential as early diagnostic biomarkers for various conditions. In this study, we developed and validated a rapid, sensitive, and accurate UPLC-MS/MS method for quantifying eight types of modified nucleosides (N1-methyladenosine (m1A), N6-methyladenosine (m6A), 5-methyluridine (m5U), 5-taurinomethyl-2-thiouridine (τm5s2U), 5-methylcytidine (m5C), 2'-O-methylcytidine (Cm), N1-methylguanosine (m1G), and N7-methylguanosine (m7G) in human urine. Using the method, we measured the urinary concentrations of m1A, m6A, m5U, τm5s2U, m5C, Cm, m1G, and m7G in a total of 21 control individuals and 23 patients diagnosed with diabetic retinopathy (DR). Cm levels showed promise as a diagnostic marker for diabetic retinopathy (DR), with a significant value (P < 0.01) and an AUC of 0.735. Other modified nucleosides also exhibited significant differences within specific subpopulations. As non-proliferative diabetic retinopathy (NPDR) signifies the latent early stage of diabetic retinopathy, we developed a multivariate linear model that integrates patients' sex, age, height, and urinary concentration of modified nucleosides which aims to predict and differentiate between healthy individuals, NPDR patients, and proliferative diabetic retinopathy (PDR) patients. Encouragingly, the model achieved satisfactory accuracy rates: healthy (81%), NPDR (75%), and PDR (80%). Our findings provide valuable insights into the development of an early, cost-effective, and noninvasive diagnostic approach for diabetic retinopathy.

Automated Identification of Modified Nucleosides during HRAM-LC-MS/MS using a Metabolomics ID Workflow with Neutral Loss Detection.

The role of post-transcriptional modification in biological processes has been an ongoing field of study for several decades. Improvements in liquid chromatography platforms and mass spectrometry instrumentation have resulted in the enhanced identification, characterization, and quantification of modified nucleosides in biological systems. One consequence of the rapid technological improvements in the analytical acquisition of modified nucleosides has been a dearth of robust data processing workflows for analyzing more than a handful of samples at a time. To improve the utility of LC-MS/MS for batch analyses of modified nucleosides, a workflow for automated nucleoside identification has been developed. We adapted the Thermo Fisher Scientific metabolomics identification software package, Compound Discoverer, to accurately identify modified nucleosides from batch LC-MS/MS acquisitions. Three points of identification are used: accurate mass from a monoisotopic mass list, spectral matching from a spectral library, and neutral loss identification. This workflow was applied to a batch (n = 24) of urinary nucleosides, resulting in the accurate identification and relative quantification of 16 known nucleosides in less than 1 h.

Graphene as adsorbent for highly efficient extraction of modified nucleosides in urine prior to liquid chromatography-tandem mass spectrometry analysis

RNA modifications have been involved in numerous biological processes, and aberrations of these modifications are tightly associated with various diseases including cancer. Herein, we developed graphene-based solid-phase extraction and robust ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) combined with stable isotope-dilution for simultaneous enrichment and accurate determination of 17 modified nucleosides in human urine. We found graphene could effectively adsorb various modified nucleosides in human urine samples. With this method, we identified and quantified these modified nucleosides in urine samples collected from lung cancer patients and healthy controls. We revealed that the levels of 12 modified nucleosides were all diminished in urine from lung cancer patients, compared with healthy controls. It is worth noting that we demonstrated, for the first time, the presence of 5,2′-O-dimethyluridine (m5Um) in human urine. Together, we established a robust analytical method for simultaneous determinations of 17 modified nucleosides in human urine, and our results revealed a close correlation between the concentrations of urinary modified nucleosides and the occurrence of lung cancer, implying the potential applications of these modified nucleosides as noninvasive biomarkers for the early detection of lung cancer. Moreover, this study will stimulate future investigations on the regulatory roles of RNA modifications in the initiation and progression of lung cancer.

Targeted quantitative metabolomics with a linear mixed-effect model for analysis of urinary nucleosides and deoxynucleosides from bladder cancer patients before and after tumor resection.

In the present study, we developed and validated a fast, simple, and sensitive quantitative method for the simultaneous determination of eleven nucleosides and deoxynucleosides from urine samples. The analyses were performed with the use of liquid chromatography coupled with triple quadrupole mass spectrometry. The sample pretreatment procedure was limited to centrifugation, vortex mixing of urine samples with a methanol/water solution (1:1, v/v), evaporation and dissolution steps. The analysis lasted 20min and was performed in dynamic multiple reaction monitoring mode (dMRM) in positive polarity. Process validation was conducted to determine the linearity, precision, accuracy, limit of quantification, stability, recovery and matrix effect. All validation procedures were carried out in accordance with current FDA and EMA regulations. The validated method was applied for the analysis of 133 urine samples derived from bladder cancer patients before tumor resection and 24h, 2weeks, and 3, 6, 9, and 12months after the surgery. The obtained data sets were analyzed using a linear mixed-effect model. The analysis revealed that concentration level of 2-methylthioadenosine was decreased, while for inosine, it was increased 24h after tumor resection in comparison to the preoperative state. The presented quantitative longitudinal study of urine nucleosides and deoxynucleosides before and up to 12months after bladder tumor resection brings additional prospective insight into the metabolite excretion pattern in bladder cancer disease. Moreover, incurred sample reanalysis was performed proving the robustness and repeatability of the developed targeted method.

Development of Optical-Based Molecularly Imprinted Nanosensors for Adenosine Detection.

Adenosine nucleoside is an important molecule in human physiology. The levels of adenosine nucleoside in urine and plasma are directly or indirectly related to diseases such as neurodegenerative diseases and cancer. In the present study, adenosine-imprinted and non-imprinted poly(2-hydroxyethyl methacrylate-methacrylic acid) (poly(HEMA-MAA)) surface plasmon resonance (SPR) nanosensors were prepared for the determination of adenosine nucleoside. First, MAA/adenosine pre-polymerization complexes were prepared at different molar ratios using adenosine as a template molecule and methacrylic acid (MAA) as a monomer, and SPR nanosensor surfaces were optimized by determining the highest imprinting factor of the chip surfaces. The surfaces of adenosine-imprinted and non-imprinted SPR nanosensors were characterized by using atomic force microscopy, ellipsometry, and contact angle measurements. Kinetic analyses were made with different concentrations in the range of 0.5-400.0 nM for the detection range with a pH 7.4 phosphate buffer solution. The limit of detection in adenosine aqueous solutions, artificial plasma, and artificial urine was determined to be 0.018, 0.015, and 0.013 nM, respectively. In the selectivity analysis of the developed nanosensors, the selectivity of adenosine SPR nanosensors in solutions at different concentrations was determined by using guanosine and cytidine nucleosides. The relative selectivity coefficients of adenosine-imprinted SPR nanosensors for adenosine/cytidine and adenosine/guanosine are 3.836 and 3.427, respectively. Since adenosine-imprinted SPR nanosensors are intended to be used in medical analysis and research, adenosine analysis has also been studied in artificial urine and artificial plasma samples.

Simultaneous Determination of Methylated Nucleosides by HILIC-MS/MS Revealed Their Alterations in Urine from Breast Cancer Patients.

RNA methylation plays a vital role in the pathogenesis of a variety of diseases including cancer, and aberrant levels of modified nucleosides in RNA were revealed to be related to cancer. Urine is a favored source for biomarker discovery due to the non-invasion to patients. Herein, we developed a sensitive hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC–MS/MS) method combined with stable isotope dilution for accurate quantification of methylated nucleosides in human urine. With this method, we successfully quantified ten methylated nucleosides in urine samples collected from healthy controls and breast cancer patients. We found N6-methyladenosine (m6A), 2′-O-methyladenosine (Am), N1-methyladenosine (m1A), N6,2′-O-dimethyladenosine (m6Am), N1-methylguanosine (m1G), 2′-O-methylguanosine (Gm), 5-methylcytidine (m5C) and 2′-O-methylcytidine (Cm) were all decreased in early-stage breast cancer patients, and a nomogram prediction model was constructed. Locally advanced breast cancer patients exhibited elevated levels of urinary 2′-O-methylated nucleosides in comparison to early-stage breast cancer patients. Together, we developed a robust method for the simultaneous determination of methylated nucleosides in human urine, and the results revealed an association between the contents of urinary methylated nucleosides and the occurrence of breast cancer, which may stimulate future studies about the regulatory roles of these methylated nucleosides in the initiation and progression of breast cancer.

Determination of adenosine and its modifications in urine and plasma from breast cancer patients by hydrophilic interaction liquid chromatography-tandem mass spectrometry

RNA modifications have been revealed to be essential in many biological activities, and their disorders are associated with various human diseases, including cancers. 2′-O-methyladenosine (Am), N1-methyladenosine (m1A), N6-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am) and N6,N6-dimethyladenosine (m62A) are important adenosine (A) modifications. The noninvasive collection of urine samples and the diverse contents of metabolites in plasma make them favored biofluids for biomarkers discovery. In this work, we established a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method to quantify these six nucleosides in urine and plasma of healthy controls and breast cancer (BC) patients. The limit of detection (LOD) for A, Am, m1A, m6A, m6Am, and m62A were 0.0025, 0.01, 0.05, 0.005, 0.005, and 0.005 nM. The results showed that the concentrations of Am, m6A, and m6Am were increased, whereas m1A was decreased in the urine of BC patients compared with the healthy controls. We also found that the level ratios of m1A/A, m6A/A, and m6Am/A were all reduced in plasma from BC patients, compared with healthy controls. Interestingly, these ratios of methylated adenosine nucleosides to adenosine in plasma could better discriminate BC patients from healthy controls, compared to the levels of these nucleosides. The present study not only suggests these modified adenosines can act as noninvasive biomarkers of BC but also will contribute to investigating the impacts of RNA methylation on the occurrence and development of BC.

BH+/MH+-matching method for discovery of cis-diol-containing modified nucleosides in urine by ribose-targeted solid phase extraction followed by dual-mass spectrometry platform identification

Profiling of new modified nucleosides from urine plays an important role in exploring biomarkers for cancer. However, limitations from the nature of the compound, bio-sample, instrumentation, and analytical method pose great challenges to achieving a comprehensive analysis of urinary nucleosides. Herein, a method of BH+/MH+-matching (BH+, protonated nucleobase ion; MH+, protonated precursor ion) was developed to discover novel modified nucleosides from human urine samples based on solid phase extraction targeted toward specific modified nucleosides combined with ultra-performance liquid chromatography coupled with dual-mass spectrometry platforms. Firstly, nucleosides containing 2,3-diol structure on ribose were effectively enriched by PBA (Phenylboronic Acid) cartridges. Secondly, a novel method, “BH+/MH+-matching” was established to achieve rapid screening of modified nucleosides. Based on the in-source fragmentation pattern of nucleosides, a series of putative modified nucleosides were rationally designed and characterized by matching the daughter ion BH+ and its parent ion MH+ in UPLC-MSE spectra. Finally, as a complement to UPLC Q-TOF/MS, UPLC Q-Trap/MS was employed to validate the structure of putative compounds by MRM-IDA-EPI mode. Using the strategy, 12 new cis-diol-containing nucleoside analogs were successfully characterized, which were formed by modified base (m1A, m6A, m2,2,7G, ac4C) and modified ribose containing C5′-O-formylation or C5′-O-methylation. Taken together, the results demonstrated our strategy could efficiently support the rapid discovery of cis-diol-containing nucleosides with modifications on either ribose or base moiety (or both), which exhibited a promising perspective in the future application of biochemical analysis and clinical diagnosis.

HILIC-MS/MS for the Determination of Methylated Adenine Nucleosides in Human Urine.

N6-methyl-2'-deoxyadenosine (m6dA) is a newly discovered DNA epigenetic mark in mammals. N6-methyladenosine (m6A), 2'-O-methyladenosine (Am), N6,2'-O-dimethyladenosine (m6Am), and N6,N6-dimethyladenosine (m62A) are common RNA modifications. Previous studies illustrated the associations between the aberrations of these methylated adenosines in nucleic acids and cancer. Herein, we developed Fe3O4/graphene-based magnetic dispersive solid-phase extraction for the enrichment and hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS/MS) for the measurements of m6dA, m6A, Am, m6Am, and m62A in human urine samples. We found that malic acid could improve the HILIC-based separation of these modified nucleosides and markedly enhance the sensitivity of their MS detection. With this method, we accurately quantified the contents of these modified adenine nucleosides in urine samples collected from gastric and colorectal cancer patients as well as healthy controls. We found that, relative to healthy controls, urinary m6dA and Am levels are significantly lower for gastric and colorectal cancer patients; while gastric cancer patients also exhibited lower levels of urinary m6A, the trend was opposite for colorectal cancer patients. Together, we developed a robust analytical method for simultaneous measurements of five methylated adenine nucleosides in human urine, and our results revealed an association between the levels of urinary methylated adenine nucleosides and the occurrence of gastric as well as colorectal cancers, suggesting the potential applications of these modified nucleosides as biomarkers for the early detection of these cancers.

Circadian and chemotherapy-related changes in urinary modified nucleosides excretion in patients with metastatic colorectal cancer

Urinary levels of modified nucleosides reflect nucleic acids turnover and can serve as non-invasive biomarkers for monitoring tumour circadian dynamics, and treatment responses in patients with metastatic colorectal cancer. In 39 patients, median overnight urinary excretion of LC-HRMS determinations of pseudouridine, was ~ tenfold as large as those of 1-methylguanosine, 1-methyladenosine, or 4-acetylcytidine, and ~ 100-fold as large as those of adenosine and cytidine. An increase in any nucleoside excretion after chemotherapy anticipated plasma carcinoembryonic antigen progression 1–2 months later and was associated with poor survival. Ten fractionated urines were collected over 2-days in 29 patients. The median value of the rhythm-adjusted mean of urinary nucleoside excretion varied from 64.3 for pseudouridine down to 0.61 for cytidine. The rhythm amplitudes relative to the 24-h mean of 6 nucleoside excretions were associated with rest duration, supporting a tight link between nucleosides turnover and the rest-activity rhythm. Moreover, the amplitude of the 1-methylguanosine rhythm was correlated with the rest-activity dichotomy index, a significant predictor of survival outcome in prior studies. In conclusion, urinary excretion dynamics of modified nucleosides appeared useful for the characterization of the circadian control of cellular proliferation and for tracking early responses to treatments in colorectal cancer patients.

BH+/MH+-Matching Method for Discovery of Cis-Diol-Containing Modified Nucleosides in Urine by Ribose-Targeted Solid Phase Extraction Followed by Dual-Mass Spectrometry Platform Identification

BH+/MH+-Matching Method for Discovery of Cis-Diol-Containing Modified Nucleosides in Urine by Ribose-Targeted Solid Phase Extraction Followed by Dual-Mass Spectrometry Platform Identification

ESI-CID spectral characterization and differentiation of the cross links of thymine formed by one electron oxidation with SO4●-

The analysis of urinary nucleosides is becoming a very good tool in the diagnosis of diseases like AIDS and cancer and consequently the identification of modified nucleosides using mass spectrometry is an area of utmost importance. In this context, there is a high relevance in the understanding of the mechanism of collisionally induced dissociation (CID) of these heterocyclics. The present work characterizes and differentiates the cross-linked products formed by one electron oxidation of thymine (T). Three dimers A, B and C, were analyzed, out of which the dimer A is a C5–C5’ cross link of two T molecules containing a fused tetrahydrofuran ring, while B and C are N–C cross linked products. B and C showed very similar fragmentation pattern but that of A was different. The differentiation between these three were made by monitoring two characteristic peaks, the water loss peak ([M+H]+) and the protonated T fragment ([T + H]+). The probable mechanism of formation of these fragments and their CID mechanism in both positive and negative ionization modes are also explained. The neutral losses of NH3, H2O and NHCO were the prominent mechanism in the positive mode, while in the negative mode, only NHCO and CO losses were observed.

Reduced levels of modified nucleosides in the urine of autistic children. Preliminary studies

The aim of this study was to investigate and compare the levels of concentration of modified nucleosides in the urine of autistic and healthy children. The compounds have never been analyzed before. The levels of nucleosides in the urine of both groups were determined by validated high performance liquid chromatography coupled to mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode. Chromatographic separation was achieved with HILIC column and tubercidin was used as the internal standard for the quantification of urinary nucleosides. The within run accuracy and precision ranged from 89 to 106% and from 0.8% to 4.9%, respectively. Lower levels of O-methylguanosine, 7-methylguanosine, 1-methyladenosine, 1-methylguanine, 7-methylguanine and 3-methyladenine in the urine of 22 children with autism, aged 3 to 16 were observed. The differences were not observed in 20 healthy volunteers, in a similar age group. These findings show that modified nucleosides there are metabolic disturbances and nutritional deficiencies in autistic children.

Evaluation of bisphenol A exposure induced oxidative RNA damage by liquid chromatography-mass spectrometry

Highlighted evidence suggests the possible implication of bisphenol A (BPA) exposure on a variety of biological functions, such as DNA damage. Similar to DNA, exposed to BPA may also have potential risks to RNA damage due to its induction of reactive oxygen species. However, there are no related research reports about such health risks of BPA. Therefore, this work tried to investigate the BPA exposure induced oxidative RNA damage by detecting urinary nucleosides, the end-products of RNA metabolism. An ultra-high performance liquid chromatography-Orbitrap mass spectrometry method was applied to selectively and sensitively determine urinary nucleosides. As a result, 66 nucleosides were identified and the effects of BPA exposure on these nucleosides in rat urine samples were evaluated. The nucleosides showed different changing tendency along with different exposure dose of BPA. The strongest effect was observed in high does-exposure rats, indicating dose-response relationship between BPA-treatment and urinary nucleosides. Significant change of some nucleosides, including 8-oxoguanosine, was observed in the high-dose exposure group, suggesting obvious RNA damage to rats. To the best of our knowledge, it is the first study about the RNA damage induced by BPA exposure. The results provided a new perspective on the toxic effects of BPA exposure.

“Pseudostationary Ion-Exchanger” Sweeping as an Online Enrichment Technique in the Determination of Nucleosides in Urine via Micellar Electrokinetic Chromatography

The presented study aims to develop a new online enrichment strategy [“pseudostationary ion-exchanger” (PSIE) sweeping] for the analysis of highly hydrophilic nucleosides in urine samples with a special focus on the fundamental aspects regarding the enrichment process itself. In the first method, we employ the ionic liquid (IL)-type surfactant 1-tetradecyl-3-methylimidazolium bromide (C14MImBr) as micelle forming agent under alkaline pH conditions. It is shown that maximum enrichment efficiency can be obtained by keeping the retention factors very high within the sample zone and very low within the background electrolyte (BGE) while maintaining a sufficient resolution for the studied analytes. With this method, detection limits as low as 0.1 µg mL−1 are obtained for all analytes studied. For the nucleosides, adenosine and cytidine, a second method is developed using sodium dodecyl sulfate (SDS) as micelle forming agent under acidic pH conditions. In addition, we investigate the effect of replacing ionic buffering constituents with a zwitterionic/isoelectric buffering compound (aspartic acid) with regard to separation and enrichment efficiency. With the second method, the achieved limits of detection are as low as 0.1 µg mL−1 for Ado and 0.2 µg mL–1 for Cyd. The applicability of the two complementary methods to the analysis of the nucleosides under investigation is shown for blank and spiked human urine samples after their extraction using the commercially available phenylboronate affinity gel.

Urinary Nucleosides and Deoxynucleosides.

Urinary nucleosides and deoxynucleosides are mainly known as metabolites of RNA turnover and oxidative damage of DNA. For several decades these metabolites have been examined for their potential use in disease states including cancer and oxidative stress. Subsequent improvements in analytical sensitivity and specificity have provided a reliable means to measure these unique molecules to better assess their relationship to physiologic and pathophysiologic conditions. In fact, some are currently used as antiviral and antitumor agents. In this review we provide insight into their molecular characteristics, highlight current separation techniques and detection methods, and explore potential clinical usefulness.

Using UHPLC Q-Trap/MS as a complementary technique to in-depth mine UPLC Q-TOF/MS data for identifying modified nucleosides in urine.

Modified nucleosides, metabolites of RNA, are potential biomarkers of cancer before the appearance of morphological abnormalities. It is of great significance to comprehensively detect and identify nucleosides in human urine for discovery of cancer biomarkers. However, the lower abundance, the greater polarity and the matrix effects make it difficult to detect urinary nucleosides. In this paper, an integrated method consisted of sample preparation followed by ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC Q-TOF/MS) detection and primary identification, then ultra-high performance liquid chromatography coupled with hybrid triple quadrupole linear ion trap mass spectrometer (UHPLC Q-Trap/MS) further identification and validation were introduced. Firstly, to enrich the nucleosides and eliminate the urine matrix effects, different sorbent materials of solid phase extraction (SPE) and the elution conditions were screened. Secondly, UPLC Q-TOF/MS was used to acquire mass data in MSE mode. The structural formulas of nucleosides in urine sample were primarily identified according to retention time, accurate mass precursor ions and fragment ions from in-house database and online database. Thirdly, the preliminary identified nucleoside structures lacking of characteristic fragment ions were verified by UHPLC Q-Trap/MS in multiple reaction monitoring trigger enhanced product ion scan (MRM-EPI) and neutral loss scan (NL). At last, phenylboronic acid (PBA)-based SPE was utilized due to its higher MS signal and weaker matrix effects under optimized extraction conditions. Fifty-five nucleosides were primarily identified by UPLC Q-TOF/MS, among which 50 nucleosides were confirmed by UHPLC Q-Trap/MS. Five nucleosides, namely 4',5'-didehydro-5'-deoxyadenosine, 4',5'-didehydro-5'-deoxyinosine, isonicotinamide riboside, peroxywybutosine and hydroxywybutosine, were found from urine for the first time. The results will expand the Human Metabolome Database (HMDB).

Preparation of a silica stationary phase co-functionalized with Wulff-type phenylboronate and C12 for mixed-mode liquid chromatography

A silica stationary phase was designed and synthesized through the co-functionalization of silica with Wulff-type phenylboronate and C12 for mixed-mode liquid chromatography applications. The as-synthesized stationary phase was characterized by elemental analysis and Fourier Transform-InfraRed Spectroscopy (FT-IR). Retention mechanisms, including boronate affinity (BA), reversed-phase (RP) and anion-exchange (AE), were involved. Retention mechanism switching was easily realized by adjustment of the mobile phase constitution. Cis-diol compounds could be selectively captured under neutral conditions in BA mode and off-line separated in RP mode. Neutral, basic, acidic and amphiprotic compounds were chromatographed on the column in RP chromatography, while inorganic anions were chromatographed in AE chromatography to characterize the mixed-mode nature of the prepared stationary phase. In addition, the RP performance was compared with an octadecyl silica column in terms of column efficiency (N/m), asymmetry factor (Af), retention factor (k) and resolution (Rs). The prepared stationary phase offered multiple interactions with analytes in addition to hydrophobic interactions under RP elution conditions. Based on the mixed-mode properties, off-line 2D-LC, for selective capture and separation of urinary nucleosides, was successfully realized on a single column, demonstrating its powerful application potential for complex samples.